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Nicotinic acetylcholine a4b2* receptors (nAChRs) are implicated in various neurodegenerative diseases and smoking addiction. Imaging of brain high-affinity a4b2* nAChRs at the cellular and subcellular levels would greatly enhance our understanding of their functional role. Since better resolution could be achieved with fluorescent probes, using our previously developed positron emission tomography (PET) imaging agent [18F] nifrolidine, we report here design, synthesis and evaluation of two fluorescent probes, nifrodansyl and nifrofam for imaging a4b2* nAChRs. The nifrodansyl and nifrofam exhibited nanomolar affinities for the a4b2* nAChRs in [3H] cytisine-radiolabeled rat brain slices. Nifrofam labeling was observed in a4b2* nAChR-expressing HEK cells and was upregulated by nicotine exposure. Nifrofam co-labeled cell-surface a4b2* nAChRs, labeled with antibodies specific for a b2 subunit extracellular epitope indicating that nifrofam labels a4b2* nAChR high-affinity binding sites. Mouse brain slices exhibited discrete binding of nifrofam in the auditory cortex showing promise for examining cellular distribution of a4b2* nAChRs in brain regions.